#METABOLOMICS WORKBENCH YumengPei_20251022_032609 DATATRACK_ID:6585 STUDY_ID:ST004308 ANALYSIS_ID:AN007170 PROJECT_ID:PR002724
VERSION             	1
CREATED_ON             	October 23, 2025, 8:45 pm
#PROJECT
PR:PROJECT_TITLE                 	The Impact of IRF7 Overexpression on the Lipidome in THP1 Cells
PR:PROJECT_SUMMARY               	The polarization of macrophages into distinct functional phenotypes, namely the
PR:PROJECT_SUMMARY               	pro-inflammatory M1 and the pro-reparative M2 states, is a critical determinant
PR:PROJECT_SUMMARY               	in immune response and disease pathogenesis. This process is underpinned by
PR:PROJECT_SUMMARY               	profound metabolic reprogramming, with the lipidome being increasingly
PR:PROJECT_SUMMARY               	recognized as a key regulator and readout of macrophage function. Interferon
PR:PROJECT_SUMMARY               	regulatory factor 7 (IRF7), a transcription factor well-known for its master
PR:PROJECT_SUMMARY               	role in type I interferon signaling, has recently been implicated in immune cell
PR:PROJECT_SUMMARY               	differentiation and metabolic regulation. However, its specific function in
PR:PROJECT_SUMMARY               	governing the lipid metabolic landscape during human macrophage polarization
PR:PROJECT_SUMMARY               	remains largely unexplored. The primary goal of this project was to define the
PR:PROJECT_SUMMARY               	specific impact of IRF7 overexpression on the lipidome of human macrophages and
PR:PROJECT_SUMMARY               	to elucidate its role in polarization. We utilized a model of THP-1-derived
PR:PROJECT_SUMMARY               	macrophages to test the central hypothesis that enforced expression of IRF7
PR:PROJECT_SUMMARY               	drives a distinct lipid remodeling program, which in turn contributes to a
PR:PROJECT_SUMMARY               	specific macrophage polarization phenotype. This study aimed to provide a
PR:PROJECT_SUMMARY               	comprehensive, untargeted lipidomic profile following IRF7 overexpression,
PR:PROJECT_SUMMARY               	thereby bridging the gap between IRF7 signaling and metabolic regulation in
PR:PROJECT_SUMMARY               	macrophages. Our lipidomic analysis successfully identified a unique and
PR:PROJECT_SUMMARY               	significant alteration in the lipid profile of IRF7-overexpressing macrophages.
PR:PROJECT_SUMMARY               	We observed specific shifts in key lipid classes, which are consistent with the
PR:PROJECT_SUMMARY               	M2-like macrophages lipidomic profile. These findings provide the first
PR:PROJECT_SUMMARY               	evidence, to our knowledge, that IRF7 is a potent regulator of lipid metabolism
PR:PROJECT_SUMMARY               	in human macrophages.
PR:INSTITUTE                     	Zhejiang University
PR:LAST_NAME                     	Pei
PR:FIRST_NAME                    	Yumeng
PR:ADDRESS                       	Wu Tong Road Number 366, Hang Zhou, Zhejiang, China
PR:EMAIL                         	0623B01@zju.edu.cn
PR:PHONE                         	(0571)87236114
#STUDY
ST:STUDY_TITLE                   	The Impact of IRF7 Overexpression on the Lipidome in THP1 Cells
ST:STUDY_SUMMARY                 	THP-1 cells were cultured in RPMI-1640 medium and subjected to lentiviral
ST:STUDY_SUMMARY                 	transduction for 48 hours. Cells were divided into two treatment groups: one
ST:STUDY_SUMMARY                 	transduced with a control lentivirus and the other with a human
ST:STUDY_SUMMARY                 	IRF7-overexpressing lentivirus. The transduction was performed at a multiplicity
ST:STUDY_SUMMARY                 	of infection (MOI) of 30 in the presence of 8 μg/mL Polybrene. Simultaneously,
ST:STUDY_SUMMARY                 	all groups were treated with PMA to induce adhesion and differentiation into
ST:STUDY_SUMMARY                 	macrophage-like cells. The experiment was conducted with six biological
ST:STUDY_SUMMARY                 	replicates (N=6) per group. Following the 48-hour treatment period, successful
ST:STUDY_SUMMARY                 	cell adhesion and morphological differentiation were confirmed by microscopic
ST:STUDY_SUMMARY                 	examination. The differentiated cells were then harvested by trypsinization. The
ST:STUDY_SUMMARY                 	resulting cell pellets were collected for subsequent metabolomic analysis, which
ST:STUDY_SUMMARY                 	aims to identify the metabolic reprogramming induced by IRF7 overexpression in
ST:STUDY_SUMMARY                 	human macrophages.
ST:INSTITUTE                     	Zhejiang University
ST:LAST_NAME                     	Pei
ST:FIRST_NAME                    	Yumeng
ST:ADDRESS                       	Wu Tong Road Number 366, Hang Zhou, Zhejiang, China
ST:EMAIL                         	0623B01@zju.edu.cn
ST:PHONE                         	(0571)87236114
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	NC_1	Treatment:Control | Sample source:THP-1 cell	Gentotype=Wild-type; RAW_FILE_NAME(Raw file name)=NC_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	NC_2	Treatment:Control | Sample source:THP-1 cell	Gentotype=Wild-type; RAW_FILE_NAME(Raw file name)=NC_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	NC_3	Treatment:Control | Sample source:THP-1 cell	Gentotype=Wild-type; RAW_FILE_NAME(Raw file name)=NC_3.RAW
SUBJECT_SAMPLE_FACTORS           	-	NC_4	Treatment:Control | Sample source:THP-1 cell	Gentotype=Wild-type; RAW_FILE_NAME(Raw file name)=NC_4.RAW
SUBJECT_SAMPLE_FACTORS           	-	NC_5	Treatment:Control | Sample source:THP-1 cell	Gentotype=Wild-type; RAW_FILE_NAME(Raw file name)=NC_5.RAW
SUBJECT_SAMPLE_FACTORS           	-	NC_6	Treatment:Control | Sample source:THP-1 cell	Gentotype=Wild-type; RAW_FILE_NAME(Raw file name)=NC_6.RAW
SUBJECT_SAMPLE_FACTORS           	-	IRF7_1	Treatment:IRF7 | Sample source:THP-1 cell	Gentotype=IRF7-overexpression; RAW_FILE_NAME(Raw file name)=IRF7_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	IRF7_2	Treatment:IRF7 | Sample source:THP-1 cell	Gentotype=IRF7-overexpression; RAW_FILE_NAME(Raw file name)=IRF7_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	IRF7_3	Treatment:IRF7 | Sample source:THP-1 cell	Gentotype=IRF7-overexpression; RAW_FILE_NAME(Raw file name)=IRF7_3.RAW
SUBJECT_SAMPLE_FACTORS           	-	IRF7_4	Treatment:IRF7 | Sample source:THP-1 cell	Gentotype=IRF7-overexpression; RAW_FILE_NAME(Raw file name)=IRF7_4.RAW
SUBJECT_SAMPLE_FACTORS           	-	IRF7_5	Treatment:IRF7 | Sample source:THP-1 cell	Gentotype=IRF7-overexpression; RAW_FILE_NAME(Raw file name)=IRF7_5.RAW
SUBJECT_SAMPLE_FACTORS           	-	IRF7_6	Treatment:IRF7 | Sample source:THP-1 cell	Gentotype=IRF7-overexpression; RAW_FILE_NAME(Raw file name)=IRF7_6.RAW
#COLLECTION
CO:COLLECTION_SUMMARY            	THP-1 cells were harvested at the logarithmic growth phase. After
CO:COLLECTION_SUMMARY            	lentivirus-mediated overexpression of IRF7, the culture medium was removed by
CO:COLLECTION_SUMMARY            	centrifugation. The cell pellets were then collected for subsequent lipidomic
CO:COLLECTION_SUMMARY            	analysis.
CO:SAMPLE_TYPE                   	Mononuclear cells
#TREATMENT
TR:TREATMENT_SUMMARY             	THP-1 cells were harvested at the logarithmic growth phase. Using lentivirus to
TR:TREATMENT_SUMMARY             	overexpress of IRF7, then add PMA to induce differentiation into macrophages.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Following IRF7 overexpression and PMA-induced differentiation into macrophages,
SP:SAMPLEPREP_SUMMARY            	the culture supernatant was completely removed. Adherent cells were harvested by
SP:SAMPLEPREP_SUMMARY            	trypsinization and the reaction was neutralized with complete culture medium.
SP:SAMPLEPREP_SUMMARY            	The cell suspension was transferred to a centrifuge tube and pelleted by
SP:SAMPLEPREP_SUMMARY            	centrifugation at 300 × g for 5 minutes at 4°C. The resulting supernatant was
SP:SAMPLEPREP_SUMMARY            	carefully aspirated and discarded. The cell pellet was then washed twice with
SP:SAMPLEPREP_SUMMARY            	ice-cold, 1X phosphate-buffered saline (PBS) to remove any residual medium
SP:SAMPLEPREP_SUMMARY            	components and metabolites that could interfere with the analysis. After the
SP:SAMPLEPREP_SUMMARY            	final wash, the PBS was thoroughly removed. The purified cell pellet was
SP:SAMPLEPREP_SUMMARY            	immediately flash-frozen in liquid nitrogen to quench all metabolic activity and
SP:SAMPLEPREP_SUMMARY            	preserve the metabolic profile intact. The frozen pellet was stored at -80°C
SP:SAMPLEPREP_SUMMARY            	until further metabolomic extraction and analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish UHPLC
CH:COLUMN_NAME                   	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)
CH:SOLVENT_A                     	60% Acetonitrile/40% Water; 10 mM ammonium acetate; 0.1% formic acid
CH:SOLVENT_B                     	10% Acetonitrile/90% Isopropanol; 10 mM ammonium acetate; 0.1% formic acid
CH:FLOW_GRADIENT                 	30% B, initial; 30% B, 2min; 43% B, 5 min；55% B, 5.1 min；70% B, 11 min；
CH:FLOW_GRADIENT                 	99%B, 16 min;30%B, 18.1min.
CH:FLOW_RATE                     	0.35 mL/min
CH:COLUMN_TEMPERATURE            	40°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF-X Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Q ExactiveTM HF mass spectrometer was operated in positive [negative] polarity
MS:MS_COMMENTS                   	mode with sheath gas :40 psi, sweep gas: 0 L/min, auxiliary gasrate: 10 L/min [7
MS:MS_COMMENTS                   	L/min], spray voltage: 3.5 kV, capillary temperature: 320°C, heater
MS:MS_COMMENTS                   	temperature: 350℃, S-Lens RF level: 50, scan range: 114–1700 m/z, automatic
MS:MS_COMMENTS                   	gain control target: 3e6, normalized collision energy: 22eV; 24 eV; 28eV [22
MS:MS_COMMENTS                   	eV；24 eV；28 eV], Injection time: 100 ms, Isolation window: 1m/z, automatic
MS:MS_COMMENTS                   	gaincontrol target（MS2）: 2e5, dynamic exclusion: 6s.
MS:MS_RESULTS_FILE               	ST004308_AN007170_Results.txt	UNITS:μg/g	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END